This proposal aims to enhance our understanding of the molecular interactions of the complexes involved in transcription initiation by RNA polymerase II. The transcription of DMA into RNA is a universal mechanism that is required of all cells for proper gene expression. Irregular initiation of transcription can lead to a variety of cellular maladies, ranging from apoptosis and aberrant differentiation to unchecked proliferation and growth. The protein complexes involved in transcription initiation are highly conserved among eukaryotes. In addition to the core RNA polymerase enzyme, the transcription machinery is also composed of associated complexes known as general transcription factors, including TFIIA, TFIIB, TFIID, TFIIF, and TFIIH. Using Saccharomyces cerevisiae, a tractable organism that can be utilized to easily obtain large quantities of these factors, this project will biochemically dissect how the general transcription factors and RNA polymerase II interact to not only form the initiation complex, but to regulate the transition to an active and processive transcription complex as well. The transcription factors, along with the core polymerase, will be purified either in recombinant forms or from the endogenous source. Taking advantage of a variety of molecular techniques, the role each factor plays during the initiation phase will be determined as well as methods of regulation in order to give us further insights as to how gene expression is coordinated.